Title: Targeting Glioblastoma Stem Cells through Epigenetic Reprogramming ABSTRACT: Glioblastoma (GBM) is the most common malignant brain tumor in adult with poor clinical outcome. There has been a lack of revolutionary improvement in treatment of this deadly disease over the past 40 years. Any treatment that can significantly prolong patients' overall survival for more than three months, which is the best achievement so far to treat GBM when using surgery, radiation therapy and temozolomide, can be considered as a success. Our long- term goal is to develop novel therapeutic strategies for this lethal disease. GBM cancer stem- like cells (CSCs) were recently prospectively isolated by several groups and showed resistance to conventional radiation therapy and chemotherapy. Targeting CSCs therapy brings a hope for brain tumor patients. The Notch signaling is a developmental signaling pathway that has been found activated in GBM CSCs. We have demonstrated that Notch inhibition deplete GBM CSCs and prolong survival in mice bearing intracranial GBM xenografts. A recent Phase I clinical trial study shows that a quarter of malignant glioma patients have stabilized disease for more than four month after Notch inhibitor treatment. However, the targets that mediate Notch regulated GBM CSC self-renewal are largely unknown. As CSCs give rise to non-CSC cells, the major difference between the two is a different level of gene expression regulated by epigenetics, such as DNA methylation and histone modification, including acetylation. Our preliminary data from microarray analysis on Notch inhibitor treated GBM neurosphere showed that an epigenetic factor Histone deacetylase 4 (DHAC4) is regulated by Notch signaling. Furthermore, our preliminary studies showed that that HDAC4 is required for GBM neurosphere propagation in vitro and in vivo. In order to develop new targets to deplete GBM CSCs, the current proposal will examine the molecular mechanism by which Notch regulates HDAC4 and its impact on GBM CSCs, and its translational application. In specific Aim #1, we will define the functional effect of different protein domains of HDAC4 on GBM CSCs. In specific Aim #2, we will define the role of Notch and HDAC4 interaction on self- renewal of GBM CSCs. In specific Aim #3, we will examine a combination of Notch and HDAC4 inhibition therapy in GBM patient-derived orthotopic xenografts (PDXs). Successes in the current proposal will not only discover how an epigenetic factor HDAC4 regulates self-renewal of GBM CSCs and improve GBM treatment, but also will have a clinical impact on cancer therapy in general.